During the first period of funding. Project 1 focused on addressing the role of progesterone receptors in mediating the cytoprotective effects of progesterone (P4). In particular. Project 1 challenged the dogma that classical progesterone receptors (PR) are the principal mediators of P4's protective effects in the brain by suggesting that recently described membrane-associated progesterone receptors may be equally important. Major findings included the observation that indeed both the classical PR and membrane-associated progesterone receptors, particularly Pgrmcl, cooperate to regulate key cytoprotective mediators, including effectors of the ERK/MAPK pathway (such as ERK1/2 and ERK5) and BDNF. In the continuation of this project, we propose to apply this knowledge to understand whether 1) there is a finite period (i.e., therapeutic window) following steroid hormone deprivation, or with age, where P4's effects on neuroprotection and key mediators of cytoprotection are maintained, and 2) if the relative abundance of PR and Pgrmcl predict sustained protective effects of P4. Further, we will determine whether specific patterns of progesterone receptor expression predict whether P4 complements or antagonizes estrogen's protective effects. Our hypothesis states that the combined expression of the classical PR and the membrane progesterone receptor, Pgrmcl, is required to maintain responsiveness of the brain to P4. Further, we propose that the relative abundance of these two receptors will also predict whether P4 complements estrogen's protective program or whether it antagonizes estrogen's effects. These hypotheses will be tested in cellular models, where the relative levels of PR and Pgrmcl can be manipulated (pharmacologically or using molecular tools), as well as in an animal model of steroid deprivation (ovariectomy), and finally, translated to a human model of ovariectomy (i.e., the surgical menopause). While the loss of estrogen's beneficial effects with increasing post-ovariectomy duration has been described, nothing is known with respect to the response to P4. Thus, the studies proposed herein will reveal a key piece of the therapeutic window puzzle by defining which progesterone receptors are critical determinants of P4's protective effects and importantly, may offer unique insight into how the therapeutic window may be expanded (i.e., through regulation of specific progesterone receptors). Together with the studies proposed in Projects 2 and 3, we expect this program of research to advance our understanding of the neurobiological basis of the critical window of therapeutic opportunity for estrogen and progesterone, a goal that could not be achieved by Project 1 alone.